Probing aromatic, hydrophobic, and steric effects on the self-assembly of an amyloid-β fragment peptide

Aromatic amino acids have been shown to promote self-assembly of amyloid peptides, although the basis for this amyloid-inducing behavior is not understood. We adopted the amyloid-β 16-22 peptide (Aβ(16-22), Ac-KLVFFAE-NH(2)) as a model to study the role of aromatic amino acids in peptide self-assembly. Aβ(16-22) contains two consecutive Phe residues (19 and 20) in which Phe 19 side chains form interstrand contacts in fibrils while Phe 20 side chains interact with the side chain of Va l18. The kinetic and thermodynamic effect of varying the hydrophobicity and aromaticity at positions 19 and 20 by mutation with Ala, Tyr, cyclohexylalanine (Cha), and pentafluorophenylalanine (F(5)-Phe) (order of hydrophobicity is Ala < Tyr < Phe < F(5)-Phe < Cha) was characterized. Ala and Tyr position 19 variants failed to undergo fibril formation at the peptide concentrations studied, but Cha and F(5)-Phe variants self-assembled at dramatically enhanced rates relative to wild-type. Cha mutation was thermodynamically stabilizing at position 20 (ΔΔG = -0.2 kcal mol(-1) relative to wild-type) and destabilizing at position 19 (ΔΔG = +0.2 kcal mol(-1)). Conversely, F(5)-Phe mutations were strongly stabilizing at both positions (ΔΔG = -1.3 kcal mol(-1) at 19, ΔΔG = -0.9 kcal mol(-1) at 20). The double Cha and F(5)-Phe mutants showed that the thermodynamic effects were additive (ΔΔG = 0 kcal mol(-1) for Cha 19,20 and -2.1 kcal mol(-1) for F(5)-Phe 19,20). These results indicate that sequence hydrophobicity alone does not dictate amyloid potential, but that aromatic, hydrophobic, and steric considerations collectively influence fibril formation.     

Influence of C-terminal α-helix hydrophobicity and aromatic amino acid content on apolipoprotein A-I functionality

The apoA-I molecule adopts a two-domain tertiary structure and the properties of these domains modulate the ability to form HDL particles. Thus, human apoA-I differs from mouse apoA-I in that it can form smaller HDL particles; the C-terminal α-helix is important in this process and human apoA-I is unusual in containing aromatic amino acids in the non-polar face of this amphipathic α-helix. To understand the influence of these aromatic amino acids and the associated high hydrophobicity, apoA-I variants were engineered in which aliphatic amino acids were substituted with or without causing a decrease in overall hydrophobicity. The variants human apoA-I (F225L/F229A/Y236A) and apoA-I (F225L/F229L/A232L/Y236L) were compared to wild-type (WT) apoA-I for their abilities to (1) solubilize phospholipid vesicles and form HDL particles of different sizes, and (2) mediate cellular cholesterol efflux and create nascent HDL particles via ABCA1. The loss of aromatic residues and concomitant decrease in hydrophobicity in apoA-I (F225L/F229A/Y236A) has no effect on protein stability, but reduces by a factor of about three the catalytic efficiencies (V_max/K_m) of vesicle solubilization and cholesterol efflux; also, relatively large HDL particles are formed. With apoA-I (F225L/F229L/A232L/Y236L) where the hydrophobicity is restored by the presence of only leucine residues in the helix non-polar face, the catalytic efficiencies of vesicle solubilization and cholesterol efflux are similar to those of WT apoA-I; this variant forms smaller HDL particles. Overall, the results show that the hydrophobicity of the non-polar face of the C-terminal amphipathic α-helix plays a critical role in determining apoA-I functionality but aromatic amino acids are not required. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

Effect of Zn ion on the structure and electronic states of Aβ nonamer: molecular dynamics and ab initio molecular orbital calculations

Aggregates of amyloid-beta proteins (Aβ) have been recognised to be intimately related to pathogenesis of Alzheimer’s disease (AD). Indeed, Aβ aggregates of various sizes from dimers to fibrils were found in the brains of AD patients, and these aggregates can be self-organised. Since abnormal accumulation of metal ions such as Zn, Cu and Fe was also observed in the brains, the association between Aβ aggregations and these metal ions has been studied widely. In the present study, to elucidate the influence of Zn ions on the stability of Aβ aggregates, we performed molecular dynamics (MD) simulations and ab initio fragment molecular orbital (FMO) calculations on the Aβ nonamers with and without Zn ions and investigated the change in its structure and electronic states induced by Zn ions at atomic and electronic levels. The MD simulations revealed that Aβ nonamer cannot keep its symmetry structure, whereas Aβ nonamer with Zn ions keeps the structure. The FMO results indicated that electrostatic interactions among the charged amino-acid residues of Aβ nonamer are significantly changed by the influence of Zn ions to stabilise Aβ nonamer. These results provide useful information for proposing novel compounds, which binds specifically to Aβ and inhibits the Aβ aggregation.     

Regulatory volume decrease in COS-7 cells at 22 °C and its influence on the Boyle van't Hoff relation and the determination of the osmotically inactive volume

Cryobiological analyses assume that the direction and rate of water movements across cell membranes and equilibrium cell volumes are determined solely by differences in the chemical potentials of intra- and extra-cellular water. A consequence of this assumption is that cells obey the Boyle van't Hoff (BvH) law which states that cell volumes are a linear function of reciprocal osmolality. Extrapolation of the BvH plot to infinite osmolality yields a quantity b, the fractional volume of the cell occupied by solids. In many cells, however, a cell volume excursion above the isotonic volume initiates an energy-requiring response that causes the swollen cells to shrink back to or towards isotonic volume. It is referred to as regulatory volume decrease (RVD). We have observed a strong RVD in COS-7 cells. If not eliminated by keeping exposure times short, this RVD produces a b that is 60% too high (0.48 vs. 0.30). These results indicate the importance of examining cells for volume regulatory mechanisms before performing measurements to determine their osmotic parameters.     

The influence of spin-labeled fluorene compounds on the assembly and toxicity of the aβ peptide

Background

The deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimer''s disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species.

Methodology/Principal Findings

To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species.

Conclusions

Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they posses both antioxidant properties and the ability to disrupt AβO species.     

The effect of α-tocopherol, α- and γ-tocotrienols on amyloid-β aggregation and disaggregation in vitro

One of the neuropathological hallmarks of Alzheimer's disease (AD)—causing neurodegeneration and consequent memory deterioration, and eventually, cognitive decline—is amyloid-β (Aβ) aggregation forming amyloid plaques. Our previous study showed the potential of a tocotrienol-rich fraction—a mixture of naturally occurring of vitamin E analogs—to inhibit Aβ aggregation and restore cognitive function in an AD mouse model. The current study examined the effect of three vitamin E analogs—α-tocopherol (α-TOC), α-tocotrienol (α-T3), and γ-tocotrienol (γ-T3)—on Aβ aggregation, disaggregation, and oligomerization in vitro. Thioflavin T (ThT) assay showed α-T3 reduced Aβ aggregation at 10 μM concentration. Furthermore, both α-T3 and γ-T3 demonstrated Aβ disaggregation, as shown by the reduction of ThT fluorescence. However, α-TOC showed no significant effect. We confirmed the results for ThT assays with scanning electron microscopy imaging. Further investigation in photo-induced cross-linking of unmodified protein assay indicated a reduction in Aβ oligomerization by γ-T3. The present study thus revealed the individual effect of each tocotrienol analog in reducing Aβ aggregation and oligomerization as well as disaggregating preformed fibrils.     

Influence of 4′-6-diamidino-2-phenylindole on the secondary structure and template activities of DNA and polydeoxynucleotides

Summary The interaction between 4-6-Diamidino-2-Phenylindole-hydrochloride (DAPI) and a variety of DNAs and synthetic polydeoxynucleotides was investigated in order to delineate the nucleic acid structural features necessary for binding. The spectra of DAPI-DNA complexes, measured at various DAPI-DNA molar ratios (r), are hypochromic relative to DNA in the region of its maximum absorption. All the curves pass through an isosbestic point at 268 nm. A new maxima appears in the region of 380–392 nm for DAPI-DNA complexes. The magnitude of the peaks in the region are directly proportional to the amount of drug present in the complex.Studies with various DNA types and synthetic polydeoxynucleotides indicate that the drug preferentially binds to dAT-rich regions of DNA. This was also confirmed by enzymatic studies. The inhibition of template action by DAPI in a purified DNA-polymerase reaction was dependent on the dAT-content of the template. The implication of these data to explain a selective binding of DAPI to mitochondrial DNA have been discussed.     

Comparison of the carbohydrate and amino acid composition of normal and S-variant α-1-antitrypsin

α-1-Antitrypsin has been isolated and purified from the serum of an individual with the Pi S phenotype whose serum contains only 50–60% as much α-1-antitrypsin as normal M-type serum. The preparation was homogeneous by the criteria of sodium dodecyl sulfate polyacrylamide gel electrophoresis and sedimentation equilibrium ultracentrifugation. When analyzed in the ultracentrifuge, the S-type α-1-antitrypsin exhibited a molecular weight of 47500 which was essentially the same as that of the M-type (47300) and the Z-type (47500) α-1-antitrypsin. The S-type α-1-antitrypsin contains 15.2% carbohydrate consisting of 16.4 residues/mol of N-acetylglucosamine, 7.8 residues/mol of mannose. 6.7 residues/mol of galactose and 7.1 residues/mol of sialic acid which is essentially the same as the carbohydrate composition of the M-type α-1-antitrypsin. In addition, M- and S-type α-1-antitrypsin have very similar amino acid compositions.     

The influence of the N-terminal region proximal to the core domain on the assembly and chaperone activity of αB-crystallin

αB-Crystallin (HSPB5) is a small heat-shock protein that is composed of dimers that then assemble into a polydisperse ensemble of oligomers. Oligomerisation is mediated by heterologous interactions between the C-terminal tail of one dimer and the core “α-crystallin” domain of another and stabilised by interactions made by the N-terminal region. Comparatively little is known about the latter contribution, but previous studies have suggested that residues in the region 54–60 form contacts that stabilise the assembly. We have generated mutations in this region (P58A, S59A, S59K, R56S/S59R and an inversion of residues 54–60) to examine their impact on oligomerisation and chaperone activity in vitro. By using native mass spectrometry, we found that all the αB-crystallin mutants were assembly competent, populating similar oligomeric distributions to wild-type, ranging from 16-mers to 30-mers. However, circular dichroism spectroscopy, intrinsic tryptophan and bis-ANS fluorescence studies demonstrated that the secondary structure differs to wild type, the 54–60 inversion mutation having the greatest impact. All the mutants exhibited a dramatic decrease in exposed hydrophobicity. We also found that the mutants in general were equally active as the wild-type protein in inhibiting the amorphous aggregation of insulin and seeded amyloid fibrillation of α-synuclein in vitro, except for the 54–60 inversion mutant, which was significantly less effective at inhibiting insulin aggregation. Our data indicate that alterations in the part of the N-terminal region proximal to the core domain do not drastically affect the oligomerisation of αB-crystallin, reinforcing the robustness of αB-crystallin in functioning as a molecular chaperone.     

The influence of amino acid side-chains on α-helix stability: S-peptide analogues and related ribonucleases S′

In order to determine the influence of amino acid side-chains on α-helix stability, in relation to the protein folding process, the coil-helix transitions of some synthetic [Orn 10]-S-peptide analogues, containing, in position 8, Phe, Tyr, Ile, Ala, cpGly² and Gly, were investigated by the technique of circular dichroism under two different sets of conditions. First, the transitions of the Speptide analogues in water/trifluoroethanol mixtures were recorded. From the pattern of the transitions and from the ellipticity values in 97% trifluoroethanol, the following increasing order of amino acids as α-helix formers was found: Gly < Tyr ≤ Phe < cpGly < Ala < Ile. This finding indicates that the conformational parameters (Chou & Fasman, 1974) of the residues in position 8 play an important but not exclusive role in α-helix stability, since the hydrophobicity of the side-chain (Nozaki & Tanford, 1971) of residue 8 exerts a strong influence. From the second approach, studying the capability of the S-peptide analogues to bind to S-protein, the following increasing order was found: (Gly, Ala) < Ile < cpGly < Tyr < Phe. This result reveals that the conformational parameters of the residues in position 8 play no role, whereas their hydrophobic character and side-chain interactions with surrounding residues in the S-protein portion are the determining binding factors. This finding explains the reason for the Phe8 invariance in RNAase A during evolution, and furnishes evidence for the relevant role of long-range interactions in the protein folding process.     

The isolation and amino acid sequence of the β- and γ-subunits of the lectin from the seeds of Dioclea Grandiflora

Although the two smaller β- and γ- subunits of the lectin from Dioclea grandiflora were clearly resolved by sodium dodecyl sulphate (SDS) gel electrophoresis, the concensus of other techniques including ultracentrifugation, isoelectric focusing in 8 M urea, size-exclusion chromatography in dissociating solvents and amino acid and sequence analysis indicated that they were similar in molecular size and that they had arisen either by a single enzymic cleavage at Asn¹¹⁸-Ser¹¹⁹ in the middle of the 237 residue-long mature α-subunit or by multiple cleavages occurring during post-translational processing of intermediates. The existence of minor forms of the β- and γ- subunits resulting from a cleavage at Asn¹²⁴-Ser¹²⁵ of the α-subunit was also recognized. The results indicated that the apparent difference in molecular size of the β- and γ-subunits deduced from SDS-gel electrophoresis could be explained by the anomalous behaviour of both subunits in this separation technique. The structural features of the D. grandiflora lectin are compared with those of concanavalin A obtained from seeds of the botanically related Canavalia ensiformis.     

Zinc-induced dimerization of the amyloid-β metal-binding domain 1-16 is mediated by residues 11-14

Analysis of complex formation between amyloid-β fragments using surface plasmon resonance biosensing and electrospray mass spectrometry reveals that region 11-14 mediates zinc-induced dimerization of amyloid-β and may serve as a potential drug target for preventing development and progression of Alzheimer's disease.     

Origin and evolution of the sulawesi macaques 2. complete amino acid sequences of seven β chains of three molecular types

Seven β chains were identified as the typical molecular types carried by the seven species of Sulawesi macaques based on isoelectric focusing and urea starch gel electrophoresis. These β chains include the β3 chains ofmaura, tonkeana, nigra, andbrunnescens, β1 chains ofhecki andochreata and β5 chain ofnigra. The results of chromatography on cation-exchange and reversed phase columns and the amino acid compositions of the tryptic peptides suggested substitutions at the 9th and 13th amino acids from the N-terminal. Sequence analyses of these seven β chains from the N-terminal to the 18th amino acid and those of purified tryptic peptides from βT3 to βT15 by Edman degradation revealed the following facts: (1) the amino acid sequences of the β3 chains carried by the four species coincided with each other and as did those of the β1 chains of the two named species; and (2) the 9th and 13th amino acids were Lys and Thr in β3, Asn and Asn in β1, and Asp and Thr in the β5 chain, respectively. These three β chains are related with each other by at least two-base changes. The evolution of the β chains of the Sulawesi macaques was inferred to be as follows. (1) The β3 chain might have been dominant β chain in the past among Sulawesi macaques, since peripheral species separately carried this chain; (2) the β1 and β5 chains might have derived from a “missing link” because of more than two-base substitutions between β3 and β1 and between β3 and β5; (3) eight other macaque species, including the lion-tailed macaque (M. silenus), bear Asn and Thr at these two positions, while the Barbary macaque (M. sylvanus) has Thr and Thr; and (4) thus, if the parsimonious rule is followed, the type with Asn-Thr is the most plausible “missing link,” since only the Asn-Thr type can combine these five β chains by minimum one-base change. Two genetic events are postulated in the evolutionary process of the Sulawesi β chains: the first Lys-Thr type (β3) was distributed over the whole island, and next Asn-Thr, the common type in other macaques, produced Asn-Asn (β1) and Asp-Thr (β5).     

Leucine biosynthesis in Alcaligenes eutrophus H16: Influence of amino acid additions on the formation of active α-isopropylmalate synthase and α-acetohydroxy acid synthase

The -isopropylmalate synthase of the chemolithoautotrophic Alcaligenes eutrophus H16 is apparently a soluble enzyme but is strongly adsorbed to cell particles in ruptured cell suspensions. This was not observed with -acetohydroxy acid synthase or threonine deaminase. The formation of these regulatory enzymes of the branched chain amino acid biosynthesis pathway generally decreased with decreased growth rates. The addition of 5 mM valine plus isoleucine with and without 5 mM threonine caused a 6.6- and a 4-fold increase, respectively, in the formation of active -isopropylmalate synthase, but caused a strong decrease in the -actohydroxy acid synthase. The level of active -isopropylmalate synthase is apparently regulated by the level of leucine; whereas, the level of the -acetohydroxy acid synthase and threonine deaminase is influenced by the presence of several amino acids. A catabolic threonine deaminase was not encountered.Abbreviations IRS -Isopropylamalate - AHA -acetohydroxy acid - TDA throninedeaminase This paper is dedicated to Professor H. G. Schlegel, University Göttingen, on the occasion of his 60th birthday. I am grateful to a great teacher and scientist, who in his unique way stimulated enthusiasm and fascination in microbiology in his students throughout the years     

Carbon nanotube inhibits the formation of β-sheet-rich oligomers of the Alzheimer's amyloid-β(16-22) peptide

Alzheimer's disease is associated with the abnormal self-assembly of the amyloid-β (Aβ) peptide into toxic β-rich aggregates. Experimental studies have shown that hydrophobic nanoparticles retard Aβ fibrillation by slowing down the nucleation process; however, the effects of nanoparticles on Aβ oligomeric structures remain elusive. In this study, we investigate the conformations of Aβ(16-22) octamers in the absence and presence of a single-walled carbon nanotube (SWCNT) by performing extensive all-atom replica exchange molecular-dynamics simulations in explicit solvent. Our simulations starting from eight random chains demonstrate that the addition of SWCNT into Aβ(16-22) solution prevents β-sheet formation. Simulation starting from a prefibrillar β-sheet octamer shows that SWCNT destabilizes the β-sheet structure. A detailed analysis of the Aβ(16-22)/SWCNT/water interactions reveals that both the inhibition of β-sheet formation and the destabilization of prefibrillar β-sheets by SWCNT result from the same physical forces: hydrophobic and π-stacking interactions (with the latter playing a more important role). By analyzing the stacking patterns between the Phe aromatic rings and the SWCNT carbon rings, we find that short ring–centroid distances mostly favor parallel orientation, whereas large distances allow all other orientations to be populated. Overall, our computational study provides evidence that SWCNT is likely to inhibit Aβ(16-22) and full-length Aβ fibrillation.     

Study of the influence of temperature on the dynamics of the catalytic cleft in 1,3-1,4-β-glucanase by molecular dynamics simulations

The dependence of some molecular motions in the enzyme 1,3-1,4-β-glucanase from Bacillus licheniformis on temperature changes and the role of the calcium ion in them were explored. For this purpose, two molecular dynamics simulated trajectories along 4 ns at low (300 K) and high (325 K) temperatures were generated by the GROMOS96 package. Several structural and thermodynamic parameters were calculated, including entropy values, solvation energies, and essential dynamics (ED). In addition, thermoinactivation experiments to study the influence of the calcium ion and some residues on the activity were conducted. The results showed the release of the calcium ion, which, in turn, significantly affected the movements of loops 1, 2, and 3, as shown by essential dynamics. These movements differ at low and high temperatures and affect dramatically the activity of the enzyme, as observed by thermoinactivation studies. The first two authors contributed equally to this work